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Innovative bioaugmentation strategies to tackle ammonia inhibition in anaerobicdigestion process-MicrobStopNH3Final Project Report

Fotidis, Ioannis; Angelidaki, Irini

Publication date:2019

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Citation (APA):Fotidis, I., & Angelidaki, I. (2019). Innovative bioaugmentation strategies to tackle ammonia inhibition inanaerobic digestion process-MicrobStopNH3: Final Project Report. Technical University of Denmark.

https://orbit.dtu.dk/en/publications/58a90e04-2331-40d1-aa47-feea2ebc8308

Innovative bioaugmentationstrategies to tackle ammoniainhibition in anaerobic digestionprocess-MicrobStopNH3Final Project Report

Ioannis A. Fotidis and Irini Angelidaki

October 2019

Innovative bioaugmentation strategies to tackle ammonia inhibition in an-

aerobic digestion process-MicrobStopNH3

Final Project Report

2019

Copyright: Reproduction of this publication in whole or in part must include the

customary bibliographic citation, including author attribution, report ti-

tle, etc.

Cover photo: The bioaugmentation concept by Ioannis A. Fotidis

Published by: DTU, Department of Environmental Engineering, Bygningstorvet, Build-

ing 115, 2800 Kgs. Lyngby Denmark

www.env.dtu.dk

ISBN: 978-87-93478-05-3

Preface

All the MicrobStopNH3 project research activities took place in the Department of

Environmental Engineering of the Technical University of Denmark by the Bioenergy

Group. During the 50 months of the project, together with the two authors of the

current report, three PhD and 16 MSc students, as well as the director and the staff

members of Lemvig biogasanlæg Amba, have participated in the project activities.

We would like to thank all the people who contributed to the completion of the Mi-

crobStopNH3 project.

Finally, we would like to thank the Energinet.dk, which under the project framework

ForskEL “Innovative bioaugmentation strategies to tackle ammonia inhibition in an-

aerobic digestion process-MicrobStopNH3” (program no. 2015-12327), has funded all

the project activities.

Kgs. Lyngby, October 2019

Ioannis A. Fotidis Irini Angelidaki

Associate Professor Professor

Content

1.1 Project details 1

1.2 Short description of project objective and results 2

1.2.1 English project description 2

1.2.2 Danish project description 2

1.3 Executive summary 2

1.4 Project objectives 4

1.5 Project results and dissemination of results 6

1.5.1 Ammonia tolerant methanogenic consortia 6

1.5.2 Bioaugmentation process development for continuous reactors 9

1.5.2.1 Mesophilic bioaugmentation 10

1.5.2.2 Thermophilic bioaugmentation 12

1.5.3 Pilot-scale verification 15

1.5.3.1 Bioaugmentation inoculum 15

1.5.3.2 Pilot-scale bioaugmentation 15

1.5.4 System analysis 16

1.5.4.1 Environmental performance 17

1.5.4.2 Economic feasibility 18

1.5.5 The students of the MicrobStopNH3 20

1.5.5.1 PhD Students 20

1.5.5.2 MSc thesis 20

1.5.5.3 Special MSc Courses 20

1.6 Dissemination activities 22

1.6.1 ISI Papers published 22

1.6.2 ISI papers under preparation 23

1.6.3 Conferences, oral presentations and posters 23

1.6.4 Utilization of project results 24

1.7 Project conclusion and perspective 26

Annex 27

Final report

1.1 Project details

Project title Innovative bioaugmentation strategies to tackle

ammonia inhibition in anaerobic digestion process

Project identification

(program abbrev. and

file)

MicrobStopNH3-12327

Name of the programme

which has funded the

project

ForskEL

Project managing com-

pany/institution (name

and address)

DTU Environment, Bygningstorvet Bygning 115,

2800 Kgs. Lyngby

Project partners

Lemvig biogasanlæg Amba

CVR (central business reg-

ister)

30060946

Date for submission 30-09-2019

MicrobStopNH3-Final Project Report 1

1.2 Short description of project objective and results

1.2.1 English project description

The MicrobStopNH3 project aimed to develop the bioaugmentation of ammonia toler-

ant methanogenic consortia, as a new technical approach to alleviate the ammonia

inhibitory effect in continuous anaerobic digesters fed with ammonia-rich substrates.

The project activities included lab-scale experiments, a pilot-scale assessment and

economic and environmental feasibility analyses of the bioaugmentation process. The

results of the project showed that it is possible and environmentally and economically

beneficial to use bioaugmentation of pure or mixed methanogenic cultures in ammo-

nia inhibited continuous reactors, to improve instantly the methane production be-

tween 13 and 40%, under both mesophilic and thermophilic conditions.

1.2.2 Danish project description

MicrobStopNH3 projektet sigtede mod at udvikle bio-augmentering (biologisk beri-

gelse med bestemte mikrooorganismer) af ammoniak-tolerante metanogene grupper

som en ny metode for at afværge den ammoniakhæmmende virkning i kontinuerlige

anaerobe reaktorer der behandler ammoniakrige substrater. Projektaktiviteterne la-

boratorie- og pilotskalaforsøg og økonomisk og miljømæssige analyser af processen.

Resultaterne af projektet viste, at det er teknisk muligt samt miljømæssigt fordelag-

tigt at anvende bio-aubmentering med rene eller blandede metanogene kulturer i

ammoniak-hæmmede biogas reaktorer. Efter tilsægning af mikrorganismerne blev

metan produktionen straks forbedret med ca. 13 - 40%, under såvel mesofile som

termofile forhold.


1.3 Executive summary

Every year the agricultural and the food industrial sectors produce vast amounts of

ammonia-rich organic wastes, which require the development of a specific, efficient

and sustainable treatment. One of the most promising and effective could be anaer-

obic digestion as it provides energy (methane) and a digestate with high nutrient

levels, which can be used as fertilizer. Unfortunately, ammonia-rich substrates are

well known to inhibit this process; it is estimated that many full-scale biogas reactors

in Denmark and worldwide are seriously affected by ammonia toxicity constantly

loosing up to 1/3 of their methane producing potential. It has been widely proven

that ammonia is mainly inhibiting the aceticlastic methanogenic pathway, while the

syntrophic acetate oxidation pathway followed by hydrogenotrophic methanogenesis

is more robust to ammonia toxicity. It is therefore reasonable to assume that the

preferential use of ammonia tolerant hydrogenotrophic methanogenic consortia could

provide a new solution to alleviate the ammonia inhibitory effect in AD process. The

MicrobStopNH3 project proposed bioaugmentation as a new technical approach to

establish these ammonia tolerant consortia in continuous digesters operating under

ammonia inhibitory pressure. Based on that, during the project this new technology

(bioaugmentation) in biogas reactors for overcoming/avoiding ammonia inhibition

was developed. The project besides many lab-scale experiments included a pilot-

scale assessment and economic and environmental feasibility analyses of the bioaug-

mentation process. The main results of the project included:

The identification and enrichment of more than ten (mesophilic and thermophilic)

ammonia tolerant methanogenic cultures that subsequently were used as bio-

augmentation inocula.

The assessment of pure and complex ammonia tolerant methanogenic commu-

nities as bioaugmentation inocula and the identification of their strengths and

weaknesses under different operation conditions.

The identification of the crucial role of hydrogenotrophic methanogens in the

success of the bioaugmentation process under extreme ammonia conditions and

the proposal of the biochemical mechanisms that dictate the process.

The definition of the “critical biomass” (minimum amount) of at least 2% v/v

bioaugmentation inoculum for a successful bioaugmentation process.

The immediate improvement of methane production between 13 and 40%, in

lab-scale continuous reactors, fed with ammonia-rich substrates, after bioaug-

mentation with pure or mixed cultures at mesophilic and thermophilic conditions.

The identification of the optimal environmental and operational conditions in or-

der to enhance the effect of the identified inocula on ammonia-rich substrates.

The development of a realistic strategy for the upscale of bioaugmentation to a

pilot-scale reactor, which yielded to an immediate methane production improve-

ment of ≈40%, compared to the inhibited period.

The clearly demonstrated positive outlook on the climate change and emissions

avoidance, due to substituted energy production in both assessed counties (i.e.

Denmark and Italy), of the bioaugmentation system analyses.

We expect that the results of the MicrobStopNH3 project can be further utilised in

full-scale reactors that are using, or they will use ammonia-rich substrates. In order

to achieve this, the readiness level of the developed technology must be moved pass

the pilot-scale assessment and create a commercially applicable process. This com-

mercially available bioaugmentation technology is the next major goal of the re-

searchers involved in the MicrobStopNH3 project as well as other researchers around

the world that they have included bioaugmentation in their research activities.


1.4 Project objectives

MicrobStopNH3 was aiming to develop an innovative, efficient and robust bioaugmen-

tation technology to counteract ammonia toxicity effect in anaerobic digesters, en-

hancing biogas production up to 35%. The project had the following sub-objectives:

Identify the best ammonia tolerant methanogenic consortia for a robust bioaug-

mentation process under mesophilic and thermophilic conditions.

Define the minimum necessary amount of ammonia tolerant methanogenic inoc-

ulum (“critical biomass”) for successful bioaugmentation in continuous stirred

tank reactors (CSTR).

Define the operational and environmental parameters to enhance the bioaug-

mentation efficiency of the identified inocula.

Validate the developed bioaugmentation protocol and applying it to a pilot-scale

reactor suffering from ammonia toxicity.

As it is analytically presented in the following sections (i.e. 1.5, 1.6, 1.7 and Annex)

of the current report, all the main objectives of the project were completed success-

fully. There was only one major (partially unexpected) problem during the completion

of the project, which was the technical preparation and the controlled operation of

the pilot-scale reactor. This led to a 14 months extension of the project's timeframe,

which allowed us to address all the technical problems of the pilot-scale reactor. After

the pilot-scale reactor became operational, the optimum bioaugmentation protocol

that was developed during the previous project activities, was applied. Overall, the

different WPs of the project have been completed and as shown in the Gantt diagram

(Table 2).

Table 2. MicrobeStopNH3 Gantt diagram

Activities 2015 2016 2017 2018 2019

Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3

Ammonia tolerant methano-

genic consortia

Bioaugmentation process de-

velopment for continuous fully

mixed reactors

Pilot-scale verification

System analysis

The milestones of the project were:

M1 Robust mesophilic and thermophilic ammonia tolerant bioaugmentation inocula.

M2 Report for optimal bioaugmentation conditions (inoculum, temperature, VFA,

ammonia, OLR, HRT).

M3 A complete bioaugmentation protocol to establish ammonia tolerant methano-

gens in large-scale CSTR reactors.

M4 Evaluation report of the bioaugmentation process technology.

All the four milestones that were described in the project have been fulfilled success-

fully. The specific documented project activity that corresponds to each one of the

milestones are indicated with the milestone abbreviation (M1-M4) in the students

(subsection 1.5.6) and dissemination activities (section 1.6) of the project.


The deliverables of the project (i.e. 4-5 ISI publications and 3 conference contribu-

tions) were exceeded by at least 3-fold since 12 published ISI papers and 12 confer-

ence contributions (section 1.6, dissemination activities) were produced. Additionally,

6 ISI papers with results from the project activities are currently under preparation.

Finally, it must be mentioned that, all the potential risks that were identified in the

project proposal (such as: a) The ability to use new mixed ammonia tolerant meth-

anogens as mesophilic and thermophilic bioaugmentation inocula. b) The identifica-

tion of the “critical biomass” of ammonia tolerant methanogenic inoculum for suc-

cessful bioaugmentation. c) The identification of the optimal environmental condi-

tions for successful bioaugmentation under thermophilic conditions. d) The successful

bioaugmentation of continuous reactors under thermophilic conditions), were ad-

dressed successfully during the project, and did not affect the completion of the pro-

ject.


1.5 Project results and dissemination of results

The MicrobStopNH3 project had four main research activities and their most signifi-

cant results are presented in the current section. Nonetheless, since it was not pos-

sible to present analytically all the project activities and their results, a brief presen-

tation of all the documented experimental activities that performed during the project

are included in the "1.6 Dissemination activities" and in the "Annex" sections of the

current report.

It must be stated that this project was not aiming at any commercial results since it

was a novel technology development and thus no turnover, exports or employment

was part of the results. However, if the developed and verified MicrobStopNH3 tech-

nology is being used in full-scale conditions, we expect great economic benefits for

the existing biogas sector, which will lead to more biogas plants to be built and thus

more people to be employed.

1.5.1 Ammonia tolerant methanogenic consortia

The main aim of the current research activity was to create and identify ammonia

tolerant mesophilic and thermophilic methanogenic consortia that could be used as

bioaugmentation inocula. In order to fulfil this aim, we had to overcome the lack of

appropriate technologies to create ammonia tolerant methanogenic consortia. There-

fore, we assessed three different cultivation methods (i.e. batch, fed-batch and

CSTR) to acclimatise methanogenic consortia to high ammonia levels. In order to

evaluate the effect of temperature in the different acclimation processes both meso-

philic (37°C) and thermophilic (53°C) inocula were used. To evaluate the three ac-

climation processes methane production efficiency, incubation time, TAN/FAN (total

ammonium nitrogen/free ammonia nitrogen) levels achieved and methanogenic ac-

tivity, were used as criteria. Finally, both stepwise and direct exposure of methano-

gens to ammonia during batch cultivation, were tested during the batch experimental

assay (Figure 1).

Figure 1. The overall all acclimatization concept.

The methane production in all batch reactors reached the theoretical value through

different incubation times (Figure 2). During direct-exposure acclimation method, the

incubation time was prolonged alongside the ammonia levels, due to the longer lag

phases. The stepwise acclimation method shortened the incubation time within each

individual ammonia level both under mesophilic and thermophilic condition compared

to direct exposure. However, even though the shorter incubation time within each

individual acclimation step, the stepwise exposure still took more time to acclimatise

the consortia to high ammonia levels than the direct method. The incubation time for

the highest ammonia levels was 78 and 91 days (Fig. 2A and Fig. 2B) under direct-


exposure for mesophilic and thermophilic condition, respectively, while it was 125

and 158 days under stepwise-exposure (Fig. 2C and Fig. 2D). This was probably due

to the accumulation of lag phases during every single step of the stepwise exposure.

On the other hand, it was reported that direct-exposure, results in higher diversity

of methanogens compared to stepwise acclimation, which may provide a better

chance for the methanogens to adapt to the high ammonia environment. Therefore,

based on the results from the current study, the direct-exposure acclimation method

seems to be preferable than the stepwise-exposure, with respect to methane pro-

duction and especially the incubation time.

Figure 2. Methane production of batch acclimation method, (A) direct-exposure a mesophilic

condition, (B) direct-exposure at thermophilic condition, (C) stepwise-exposure at mesophilic

condition, (D) stepwise-exposure at thermophilic condition.

Methane production for the fed-batch reactors followed the theoretical methane pro-

duction with only small fluctuations and finally reached a high TAN concentration of

6.56 and 6.32 g NH4+-N L-1 for mesophilic and thermophilic conditions, respectively

(Figure 3). Even though a gas production delay was seen at 4.32 g NH4+-N L-1, the

reactor recovered immediately and at the end of the experiment, methane production

was more than 83% of the theoretical expected for both fed-batch reactors. This

result clearly indicated a stable AD process without any ammonia inhibition. Inter-

estingly, the final FAN concentration was more than 1600 mg NH3-N L-1in the ther-

mophilic reactor. The successful acclimatisation to these extremely high FAN levels

at such a short time frame (64 days) has never been reported before. The initial

inocula were successfully acclimatised to 6.56 and 6.32g NH4+-N L-1 under mesophilic

and thermophilic condition, respectively. During the whole process, no instability due

to high ammonia was observed.

Figure 3. Methane accumulation at (A) mesophilic and (B) thermophilic fed-batch reactors.


Both CSTR reactors did not pass the second acclimation step (4.56 g NH4+-N L-1 for

mesophilic and 4.32 g NH4+-N L-1 for thermophilic) due to strong ammonia inhibition.

An ammonia induced “inhibited steady state” was established, with a methane yield

of around 30% of the theoretical, until the end of the experiment (Figure 4). The low

methane yield indicated that methanogens were experiencing strong inhibition from

the ammonia. The main reason for the failure of both CSTR reactors could be the

washout of methanogenic communities, which resulted in the loss of ammonia toler-

ant methanogens that were in low abundance in the initial inocula. Therefore, it can

be concluded that CSTR reactors, even if they are successful, they are not suitable

for the acclimation of ammonia-tolerant methanogenic consortia in a realistic

timeframe that will allow them to be used as bioaugmentation inocula. However, if

time is not an issue, then it could be possible to use longer HRTs and slower exposure

to higher ammonia levels to efficient acclimatise methanogenic inocula, as we have

also shown in other acclimatisation experiments contacted during the MicrobStopNH3

project.

Figure 4. Methane yield at (A) mesophilic and (B) thermophilic CSTR reactors

The activity test (Figure 5) showed that hydrogenotrophic methanogens were signif-

icantly more active than aceticlastic methanogens among most of the acclimation

methods at higher ammonia levels.

Figure 5. SMA test results of mesophilic batch direct-exposure (MBD), mesophilic batch step-

wise-exposure (MBS), mesophilic fed-batch (MFB), mesophilic CSTR (MCS), thermophilic batch

direct-exposure (TBD), thermophilic batch stepwise-exposure (TBS), thermophilic fed-batch

(TFB) and thermophilic CSTR (TCS).

Aceticlastic activity was higher only in mesophilic batch reactors, with no significant

difference between direct and stepwise acclimation approach. This therefore means

that aceticlastic had higher growth rates compared to the hydrogenotrophic meth-

anogens, under the same growth conditions. Thus, the higher aceticlastic activity


could be explained by the low FAN levels (210 mg NH3-N L-1) during the mesophilic

batch acclimation that did not affect the growth rates of the aceticlastic methanogens.

Thus, based on the activity test results and the FAN levels, hydrogenotrophic meth-

anogens had higher activity under high FAN levels (>540 mg NH3-N L-1), while aceti-

clastic methanogens were more active at low FAN levels (<210 mg NH3-N L-1).

Finally, the evaluation (Table 2) of the three different acclimatization methods, based

on the chosen assessment criteria (i.e. methanogenic activity, methane production

efficiency, incubation time and ammonia levels achieved), shown that fed-batch was

the best acclimation method compared to batch and CSTR methods, under mesophilic

and thermophilic conditions. In the current acclimatization experiment, the CSTR ac-

climation method yielded only 30% production efficiency compared to the theoretical,

while at the same time more than 83% was achieved in batch and fed-batch reactors.

Table 2. Comparison between the three different acclimation methods

Among the other acclimation methods, the shortest incubation period was achieved

together with the highest methanogenic activity and the highest FAN levels, with fed-

batch reactors for both mesophilic and thermophilic conditions. Specifically, the FAN

levels and the maximum methanogenic activity of the mesophilic fed-batch were

164% and 138% higher than batch direct-exposure method, respectively. The ther-

mophilic fed-batch acclimation method had the highest FAN levels and activity, but

also had more than 40% shorter incubation time compared to batch acclimation

methods. Therefore, based on the comparison of incubation time, methanogenic ac-

tivity, production efficiency and ammonia levels between the different acclimation

methods, it seems that fed-batch could be the acclimation method that will supply

the required ammonia-tolerant bioaugmentation inocula in the future.

In conclusion, the outcome of this process provided mesophilic and thermophilic

methanogenic communities that were capable to generate high methane yields de-

spite high concentration of ammonia and been used, together with other cultures as

bioaugmentation inocula in the following research activities of the project.

1.5.2 Bioaugmentation process development for continuous reactors

The main aim of the current research activity was to operate lab-scale CSTR reactors

under high ammonia conditions testing the best ammonia tolerant methanogenic cul-

tures as bioaugmentation inocula. Many experiments were performed during the Mi-

crobStopNH3 project, which yielded 12 published and at least 6 under preparation ISI

papers. The results of two of those experiments are presented in the current report

that collectively cover all the main experimental challenges/parameters described in

the proposal (e.g. mesophilic and thermophilic conditions, pure and mixed strains,

different real-life substrates, etc.). Finally, based on the experimental findings, the

basic microbiological and biochemical mechanisms that guarantee a successful bio-

augmentation process are pinpointed.


1.5.2.1 Mesophilic bioaugmentation

In the first bioaugmentation study, hydrogenotrophic Methanoculleus bourgensis was

bioaugmented in a mesophilic, ammonia-inhibited CSTR reactor fed mainly with mi-

croalgae, at extreme ammonia levels (i.e. 11 g NH4+-N L-1). Immediately after bio-

augmentation, methane production increased and reached 236 CH4 g-1 VS establish-

ing a new steady state (Figure 6). Compared to the baseline, 28% increase in me-

thane yield was achieved after bioaugmentation, which demonstrated a successful

strategy in recovering methane production from ammonia inhibited reactor. However,

after the instability, the reactor entered a new steady state until the end of the ex-

periment with an average production of 242 CH4 g-1 VS, which was more than 31%

higher than the baseline. Overall, after bioaugmentation, the reactor showed a sig-

nificantly higher methane production efficiency compared to the inhibited steady

state, and for the first time was demonstrated that bioaugmentation can alleviate

ammonia inhibition of AD process fed with protein-rich, 3rd generation biomass.

Figure 6. Methane yield, TAN and FAN levels throughout the experimental period

The VFA levels significantly decreased from more than 5 g L-1 to around 1 g L-1 after

bioaugmentation (Figure 7), which coincided with the increase of methane yield dis-

cussed above. It seems that the decreased VFA levels due to bioaugmentation could

mitigate the ammonia-VFA synergistic inhibition, thus resulted in a higher methane

production. At the same time, the pH increased slightly after bioaugmentation, which

might be due to the VFA consumption, but it remained within the optimal pH range

(6.5-8.5) of AD process.

Figure 7. VFA and pH variations of the CSTR reactor

To identify the microbial dynamics, three samples were taken before the bioaugmen-

tation, immediately after the bioaugmentation and at the end of the experiment,


respectively. Methanobrevibacter acididurans, Methanosarcina soligelidi and Meth-

anoculleus bourgensis were the three most abundant methanogens in all the samples

(Figure 9). The bioaugmented M. bourgensis was found in all the samples, however,

its relative abundance with respect to the archaea community increased from 3.6%

before bioaugmentation to 6.7% after bioaugmentation. Although M. bourgensis was

not the dominant methanogen, it seems that it was capable to drive the methano-

genic microbiome towards a more efficient methanogenesis process. This has been

described as a “microbiological domino effect”, where the bioaugmented culture,

even at a relative low abundance, is sufficient to drive the establishment of a new

community. In fact, the bioaugmented M. bourgensis in this study triggered microbial

community changes. The most possible explanation for the changes that M. bourgen-

sis triggered was that reduced the hydrogen partial pressure by consuming hydrogen,

which created thermodynamically favourable conditions for VFAs degradation, such

as acetate and propionate due to their sensitivity to hydrogen. The reduced VFA levels

further mitigated the synergistic inhibition of ammonia and VFA, and allowed the

growth of other methanogens, such as aceticlastic M. soligelidi. As a result, immedi-

ately after bioaugmentation, M. soligelidi increased its relative abundance from

36.3% to 66.1% and reached 86.9% at the end of the experiment.

Figure 8. Relative abundance (%) (left part) and the corresponding folds change (right part)

for the interesting archaea at different phases.

Overall, the “microbiological domino effect” triggered by the bioaugmented M. bour-

gensis was the most possible mechanism for the success of bioaugmentation in the

current experimental study. This indicated that an efficient community could be es-

tablished even with the less dominant bioaugmentation culture (Figure 9). Specifi-

cally, during the ammonia-induced inhibited steady state, both bacteria and archaea

were inhibited primarily by the high hydrogen partial pressure and the ammonia-VFA

synergistic inhibitory effect. However, the hydrogen partial pressure was reduced due

to hydrogen consumption by the bioaugmented hydrogenotrophic M. bourgensis,

thus resulted in VFAs degradation and synergistic inhibition mitigation. Therefore,

overall favourable conditions were created for the growth of ammonia-tolerant bac-

teria and archaea. Finally, as a result of the aforementioned “microbiological domino

effect”, the whole microbial community was driven towards a more efficient commu-

nity degrading protein-rich substrate, evidenced by the increased relative abundance

of ammonia-tolerant methanogens.


Figure 9. The proposed working mechanism of the successful bioaugmentation of M. bour-

gensis. Red solid and green dashed arrows present the change of the compound concentration

and the OUT’s relative abundance, respectively.

1.5.2.2 Thermophilic bioaugmentation

In the second bioaugmentation study, two bioaugmentation inocula (an enriched cul-

ture, and a mixed culture composed 50/50 by Methanoculleus thermophilus and the

enriched culture) on the recovery of ammonia-inhibited (at 5 g NH4+-N L-1) thermo-

philic continuous reactors was assessed. Three identical lab-scale CSTR reactors (Rctrl,

Renr and Rmix) fed with cattle manure were used in the current study, operating under

thermophilic (53±1°C) conditions with HRT of 15 days, and organic loading rate

(OLR) of 2.50 g VS L-1 d-1 throughout the experiment.

Immediately after bioaugmentation, the methane production of Rmix (bioaugmenta-

tion with the mixed culture) increased by 17% compared to baseline inhibited pro-

duction and established a steady state with an average yield of 130 mL CH4 g-1 VS

(78% recovery) until the end of the experiment (Figure 11a). At the same time Renr,

which was bioaugmented with only the enriched consortium, also increased its me-

thane production with an average methane yield of 133 mL CH4 g-1 VS (80% recov-

ery). Rctrl marginally increased its methane yield from 101 to 117 mL CH4 g-1 VS at

the last HRT of the experiment, indicating a degree of adaptation of the AD microbi-

ome to the ammonia toxicity, which nevertheless it was still significantly lower com-

pared to Rmix and Renr. Thus, for the first time bioaugmentation was applied success-

fully to alleviate ammonia inhibition in thermophilic CSTR reactors since Renr and Rmix

improved the methane production efficiency by 11-13% compared to Rctrl. Another

interesting finding was that the methane production was recovered much faster in

Rmix than Renr, manifested by 15% higher overall methane production during the first

ten days after bioaugmentation. This indicates that the mixed culture was a better

bioaugmentation inoculum compared to the enriched culture, which demonstrated

the importance of hydrogenotrophic methanogens included in the mixed culture. A

possible explanation for this behaviour is that the instant hydrogen partial pressure

removal by hydrogenotrophic M. thermophilus created better thermodynamic condi-

tions for the overall AD process.


Figure 10. The performance of the three reactors at different experimental phases: a) me-

thane yield, b) total VFA levels and c) pH. P1, P2, P3, P4 stands for the four different experi-

mental phases of the experiment, respectively.

In general, the VFA accumulation of each reactor matched with the methane produc-

tion variations. Specifically, before increasing the ammonia levels, the VFA levels of

all the reactors were inside the normal threshold (<1500 mg L-1) for CSTR reactors

(Figure 11b). As expected, the VFA levels increased rapidly after increasing ammonia

levels and exceeded the 1500 mg L-1 threshold. Moreover, The VFA increase also

supports that methanogenesis was more susceptible to ammonia inhibition compared

to acetogenesis. Specifically, the VFA levels in the Rctrl stabilized between 2200 to

2700 mg L- while the VFA levels in Rmix and Renr decreased rapidly and returned below

1250 mg L-11 at the end of the experiment, demonstrating the immediate positive

bioaugmentation effect. The pH levels fluctuated between 7.85 and 8.05 before the

addition of ammonia, and decreased to lower levels varying between 7.55 and 7.75

due to the VFA accumulation after ammonia addition (Figure 11c). However, due to

the high buffer capacity of manure the pH levels remained within the optimal pH

range (6.5-8.5) for AD process.

To elucidate how the bioaugmentation cultures affected the microbial composition,

four samples were taken from each reactor before TAN increase, after TAN increase

but before bioaugmentation, immediately after bioaugmentation and at the end of

the experiment, respectively. The most dominant methanogens were always Meth-

anothermobacter sp.1 in all three reactors with relative abundance among the meth-

anogens between 60 to 83% (Figure 12), verifying a well-documented strong hy-

drogenotrophic methanogenic activity in thermophilic reactors. Moreover, the relative

abundance of Methanothermobacter sp.1 decreased significantly after the addition of

ammonia, but it increased back during up to the initial levels for reactors Rctrl and

Renr, indicating its ability to adapt at high ammonia levels. However, although Meth-

anothermobacter sp. kept being dominant, its relative abundance in Rmix decreased

significantly at the end, which could be explained by the increased abundance of

bioaugmented M. thermophilus.


Figure 11. Relative abundance shown as heat map for the interesting archaea at different

phases in: a) Rctrl, b) Renr, c) Rmix,

Overall, it seemed once again that the immediate reduction of the hydrogen partial

pressure by the bioaugmented hydrogenotrophic methanogen was the main reason

for a fast and more efficient bioaugmentation. Even though both bioaugmentation

inocula resulted in an improved reactor performance, the mixed culture had a faster

effect in recovering the methane production compared to the enriched culture, which

indicated that the hydrogenotrophic M. thermophilus was the key to a successful

bioaugmentation effect.

Figure 12. Proposed mechanisms depicting the effects of bioaugmentation on the three reac-

tors. The blue solid and dashed arrows present the conversion direction and decrease of the

compounds’ concentration, respectively, and two arrows stand for stronger effect compared to

a single arrow. The orange and green processes stand for the reactors’ performance before

and after bioaugmentation, respectively.

Based on the evaluation of all operational and microbiological data, a potential bio-

augmentation working mechanism was proposed (Figure 12). Specifically, the in-

stantly decreased hydrogen partial pressure, due to the consumption by M. ther-

mophilus in Rmix, ceased homoacetogenesis, increased SAO and created favourable

conditions (Eq. (1), (2), (4)) for VFA degradation by enhancing acetogenesis. Mean-

while, the enriched consortium (mainly aceticlastic M. thermophila) in the mixed cul-

ture also degraded acetate directly. Therefore, the VFA-ammonia synergistic inhibi-

tory effect was alleviated rapidly due to the reduction of the hydrogen partial pres-

sure, which resulted in a faster methane recovery compared to the other reactors.


When only the enriched culture was used (Renr), it mainly increased acetate degra-

dation, but it did not significantly contributed to the hydrogen partial pressure reduc-

tion. This resulted in a slower acetogenic step from other VFAs. Therefore, both strat-

egies alleviated ammonia inhibition, but the bioaugmentation only with the enriched

culture did not recover the methane production as fast as the bioaugmentation with

the mixed culture.

In conclusion, the results demonstrated that the basic mechanism driving a success-

ful and fast bioaugmentation process in ammonia inhibited CSTR reactors includes

the instant hydrogen partial pressure reduction by bioaugmented ammonia-tolerant

hydrogenotrophic methanogens that allows a thermodynamic syntrophic acetate ox-

idising process, which consequently decreases the VFA levels and thus, improves the

overall AD process. Finally, it showed for the first time that bioaugmentation with

ammonia-tolerant cultures can alleviate ammonia inhibition in thermophilic continu-

ous reactors.

1.5.3 Pilot-scale verification

In WP3, the optimal bioaugmentation inoculum and bioaugmentation protocol, which

are the outcomes of WP1 and WP2, were tested in a 500 L pilot biogas reactor using

ammonia rich substrates (i.e. organic fraction of municipal solid waste-OFMSW).

1.5.3.1 Bioaugmentation inoculum

A number of 12 batch reactors with total working volume of ≈20 L and synthetically

constructed media were used to produce the necessary bioaugmentation inoculum

volume according to optimum bioaugmentation protocol determined in WP2. At the

end of this task, a sufficient amount of ammonia tolerant inoculum (Methanoculleus

Bourgensis) was produced, in order to subsequently be bioaugmented into the pilot-

scale reactor.

Figure 13. The bioaugmentation inoculum bioaugmented in the pilot-scale reactor.

1.5.3.2 Pilot-scale bioaugmentation

The 500 L pilot-scale reactor fed with bio-pulped (mechanically pretreated) OFMSW

at mesophilic conditions (37°C) was bioaugmented (Figure 14). The pilot-scale reac-

tor had a hydraulic retention time (HRT) of 20 days and an organic loading rate (OLR)

of 3.1±0.4 g VS L-1reactor d-1. The reactor operated for more than one HRT under a

steady state before it was bioaugmented with 2% v/v the bioaugmentation inoculum.

Bioaugmentation with the ammonia tolerant inoculum took place on day 21 of the

experiment.


Figure 14. The pilot-scale reactor set up and the bioaugmentation process.

The results showed that immediately after bioaugmentation the methane production

rate was increased by more than 40% compared to the methane production rate just

before bioaugmentation (Figure 15). Additionally, after bioaugmentation the reactor

recovered between 40 and 80% the previous uninhibited methane production (no

inhibition period, Figure 15). The large range of this recovery can be attributed to the

significant variations in the day to day production of a large-scale biogas reactor due

to the changes in the homogeneity of the substrate. Nonetheless, the first ever pilot-

scale bioaugmentation application seems to be very promising and paves the way for

a future full-scale application of the MicrobStopNH3 bioaugmentation technology.

Figure 15. Methane production of the pilot-scale reactor before and after bioaugmentation.

1.5.4 System analysis

The main aim of the current research activity was to perform a technological study

of the process that will include the analysis of potentials and technological constraints

of all bioaugmentation parameters, as well as the scenario of applying bioaugmenta-

tion in commercial scale anaerobic reactors. Additionally, a specific environmental

assessment to examine environmental impacts of the technology as well as prelimi-

nary economic impacts compared to a normal scenario of digesting ammonia-rich

substrates was performed.

Therefore, an environmental and economic assessment of bioaugmentation as a

strategy to overcome ammonia inhibition on anaerobic digestion processes was con-

ducted (Figure 16). The option to employ this solution to mesophilic and thermophilic

reactors was analysed in terms of its effect on climate change and economic incomes,

based on its positive results in increase methane production registered in laboratory

experiments. In order to understand the applicability of this technology at larger

scales, besides the experimental results derived from the research activities of the

MicrobStopNH3 project, real life data for Denmark and Italy were used and compared.


Specifically, an environmental and cost assessment of bioaugmentation strategy as

compared with a plant running at “inhibited steady state” was performed with the

reference process defined in terms of process steps, material and energy flows in-

volved, timeframe (2018) and country of application (i.e. Denmark and Italy).

Figure 16. Material flow scheme for bioaugmentation system analysis

The economic analysis was performed on operational costs and revenues and it re-

sulted in a greater income for the biogas plants when bioaugmentation was uses. The

biogas plant operational temperature was also considered, with the mesophilic case

being consistently more efficient than the thermophilic one, where ammonia inhibi-

tion had a stronger impact. Overall, in terms of environmental focus, climate change

and emissions avoidance due to substituted energy production, the results showed a

positive outcome using the bioaugmentation for both countries, which was more pro-

found in the Italian case compared to Denmark. The specific results of the system

analysis are presented in the following three subsections, and the functional unit of

kg CO2 equivalents per ton of wet waste entering the biogas plant was used.

1.5.4.1 Environmental performance

The overall results for the different scenarios applied to the two reference countries

showed that the savings in terms of emissions are higher in the Italian case, where

the substitute electricity and heat are mostly from fossil sources compared to Den-

mark where more alternative energy sources are used (Figure 17).

Figure 17. Global Warming Potential results with standard deviation Denmark and Italy

(meso: mesophilic conditions, thermo: thermophilic conditions, base: baseline case with no

inhibition, inh: case in “inhibited steady state”, bio: case with bioaugmentation).

Both in the mesophilic and thermophilic operating conditions, bioaugmented biogas

reactors have significantly lower environmental impact than the reactors operating


under “inhibited steady state”. This is a promising result, especially for Italy, high-

lighting how the bioaugmentation strategy can improve the overall plant efficiency

and consequently the impact of anaerobic digestion on the environment. The effect

of the reactor working temperature is also relevant. In fact, there is a more significant

improvement from bioaugmentation in mesophilic compared to thermophilic condi-

tions due to the influence of temperature on the dissociation equilibrium of FAN, and

thus the higher inhibitory effect on the AD process.

The analysis on the relevant impact sources revealed that the emissions from biogas

losses and the avoided impacts related to electricity and heat production were po-

tential the primer contributors to the formulation of the global warming impact (Fig-

ure 18). Since these were clearly the main sources of impact, their determination

could result in major errors. The differences between the two countries were carried

in the substituted electricity and heat mix, which effected the contributions of avoided

heat production, avoided electricity production, digester and pasteurization energy

use. These were all higher in the Italian case, and the negative contributions from

substituted energy made the total Italian global warming impact lower compared to

the Danish one.

Figure 18. Global Warming impact contributions for Denmark and Italy (meso: mesophilic

conditions, thermo: thermophilic conditions, base: baseline case with no inhibition, inh: case

in “inhibited steady state”, bio: case with bioaugmentation).

1.5.4.2 Economic feasibility

The economic assessment for the different scenarios applied to the two reference

countries showed that the relative preference between the different scenarios within

each country was the same as in the environmental assessment (Figure 19).


Figure 19. Economic assessment results as net income for Denmark and Italy (meso: meso-

philic conditions, thermo: thermophilic conditions, base: baseline case with no inhibition, inh:

case in “inhibited steady state”, bio: case with bioaugmentation).

Specifically, bioaugmented reactors were yielding significantly higher revenues com-

pared to ones that running under “inhibited steady state”. Once again, the economic

benefit in the Italian based scenarios were yielding better than the Danish ones. The

final numbers followed the behaviour of methane production values. In fact the higher

the methane production, the higher the energy produced, the higher the revenues

but also the avoided impacts. Hence, it is possible to claim that the proposed bioaug-

mentation strategies would have positive effects on the environmental performance

as well as the economic revenues of ammonia inhibited biogas reactors.

Figure 20. Different contributions of the cost assessment for Denmark and Italy (meso: mes-

ophilic conditions, thermo: thermophilic conditions, base: baseline case with no inhibition, inh:

case in “inhibited steady state”, bio: case with bioaugmentation).


The analysed scenarios produced different results due to higher revenues in the cases

of higher electrical and thermal energy production from the combustion of more bio-

gas. The difference between the two countries lies on the price of electricity: since in

Denmark the price is lower, the revenues were less than the Italian ones, and con-

sequently so was the predicted net income (figure 20). Thus, it becomes clear that

the electricity price is a decisive parameter.

1.5.5 The students of the MicrobStopNH3

During the 50 months of the MicrobStopNH3, at least 19 students have participated

in the project activities. In the following paragraphs, their names and specific activi-

ties are presented. The specific student activity that corresponds to project mile-

stones is indicated with the milestones abbreviations (M1-M4).

1.5.5.1 PhD Students

Three DTU PhD students have participated in the MicrobStopNH3 project activities

and two of them have already graduated.

Miao Yan. "Powdered bioaugmentation inocula to alleviate ammonia toxicity in

anaerobic digesters" (M1-M3).

Hailin Tian. "Innovative bioaugmentation strategies to tackle ammonia inhibition

in anaerobic digestion process" (M1-M3).

Han Wang. "Innovative process for digesting high ammonia containing wastes"

(M1, M2).

1.5.5.2 MSc thesis

Seven MSc student have conducted their MSc thesis as part of the MicrobStopNH3

project activities and all of them graduated successfully. It has to be mentioned that

the MSc thesis of Arnaud Jéglot received one of the six Veolia Performance Trophies

2018.

Arnaud Jéglot. "Novel cultivation strategies of ammonia tolerant methanogenic

consortia focused on: microbial growth, density and preservability" (M2, M3).

Afroditi Panagiotou. "Bioaugmentation of ammonia tolerant methanogenic con-

sortia in continuous reactors fed with ammonia-rich substrates" (M3).

Konstantinos Kissas. "The impact of different ammonia sources on aceticlastic

and hydrogenotrophic methanogenesis" (M2).

Maria Ines Sobral Lupi Caetano. "Determination of the "critical biomass" for suc-

cessful bioaugmentation strategies in ammonia-inhibited continuous anaerobic

reactors" (M2, M3).

Konstantinos Konstantopoulos. "Optimal cultivation processes and microbial

characterization of ammonia-tolerant enriched methanogenic cultures" (M1, M2).

Panagiotis Karachalios. "The significance of the ammonia–LCFA synergetic co-

inhibition effect on the thermophilic biomethanation process" (M2).

Enrico Mancini. "Acclimation processes of ammonia tolerant methanogenic con-

sortia used as bioaugmentation inocula" (M1, M2).

1.5.5.3 Special MSc Courses

Nine special courses have been given as part of the MicrobStopNH3 project activities.

Estelle Maria Goonesekera, "Preliminary assessment of different biochar types to

alleviate ammonia inhibition in anaerobic digestion" (M2, M3).

Rosa Ferrigno, "Counteracting ammonia inhibition using bioaugmentation strat-

egies of ammonia tolerant methanogenic inocula" (M2, M3).


https://www.veolia.com/en/newsroom/news/performance-trophies-2018-students-prize-boston

https://www.veolia.com/en/newsroom/news/performance-trophies-2018-students-prize-boston

https://www.inside.dtu.dk/en/dtuinside/generelt/telefonbog/person?id=134758&tab=0

Dongdong Xie. "Combined alleviation of ammonia inhibition in anaerobic diges-

tion by trace element addition and bioaugmentation of ammonia tolerant consor-

tia"(M2, M3).

Arnaud Tristan Arjuna Jeglot. "Optimal cultivation methods of high ammonia tol-

erant methanogenic consortia" (M2).

Konstantinos Kissas. "Assessment of the effect of different ammonia sources on

the aceticlastic and hydrogenotrophic methanogenesis" (M1, M2).

Elizabeth Sembera. "Environmental and cost assessment of bioaugmentation

techniques for anaerobic digestion" (M4).

Maria Ines Sobral Lupi Caetand. "Preliminary assessment of the "critical biomass"

of ammonia tolerant methanogenic consortia used as bioaugmentation" (M2,

M3).

Konstantinos Konstantopoulos. "Preliminary microbiological assessment of am-

monia tolerant bioaugmentation inocula" (M1).

Enrico Mancini. "Anaerobic digestion in protein-rich substrate with acclimatized

inoculum" (M1).


1.6 Dissemination activities

The project participants have communicated the MicrobStopNH3 results in dozens

documented and undocumented dissemination activities including scientific manu-

scripts, posters, oral presentations, student lecture, keynote presentations etc. The

documented project dissemination activities included 12 published ISI papers (first

pages attached in the Annex), at least 6 ISI papers under preparation, at least 12

conference contributions. The specific dissemination activity that corresponds to pro-

ject milestones is indicated with the milestones abbreviations (M1-M4).

1.6.1 ISI Papers published

Tian, H., Yan, M., Treu, L., Angelidaki, I., Fotidis, I.A., (2019). "Hydrogenotrophic

methanogens are the key for a successful bioaugmentation to alleviate ammonia

inhibition in thermophilic anaerobic digesters." Bioresource technology, 122070

(M2, M3). https://doi.org/10.1016/j.biortech.2019.122070

Tian, H., Mancini, E., Treu, L., Angelidaki, I., Fotidis, I.A., (2019). Bioaugmenta-

tion strategy for overcoming ammonia inhibition during biomethanation of a pro-

tein-rich substrate. Chemosphere 231, 415-422 (M2, M3).

https://doi.org/10.1016/j.chemosphere.2019.05.140

Yan, M., Fotidis, I.A., Tian, H., Khoshnevisan, B., Treu, L., Tsapekos, P., Ange-

lidaki, I., (2019). Acclimatization contributes to stable anaerobic digestion of or-

ganic fraction of municipal solid waste under extreme ammonia levels: Focusing

on microbial community dynamics. Bioresource Technology 286, 121376 (M1).

https://doi.org/10.1016/j.biortech.2019.121376

Tian, H., Treu, L., Konstantopoulos, K., Fotidis, I.A., Angelidaki, I., (2018). 16s

rRNA gene sequencing and radioisotopic analysis reveal the composition of am-

monia acclimatized methanogenic consortia. Bioresource Technology 272, 54-62

(M1). https://doi.org/10.1016/j.biortech.2018.09.128

Tian, H., Karachalios, P., Angelidaki, I., Fotidis, I.A., (2018). A proposed mech-

anism for the ammonia-LCFA synergetic co-inhibition effect on anaerobic diges-

tion process. Chemical Engineering Journal 349, 574-580 (M2).

https://doi.org/10.1016/j.cej.2018.05.083

Tian, H., Fotidis, I.A., Mancini, E., Treu, L., Mahdy, A., Ballesteros, M., González-

Fernández, C., Angelidaki, I., (2018). Acclimation to extremely high ammonia

levels in continuous biomethanation process and the associated microbial com-

munity dynamics. Bioresource Technology 247, 616-623 (M1).

https://doi.org/10.1016/j.biortech.2017.09.148

Tian, H., Fotidis, I.A., Kissas, K., Angelidaki, I., (2018). Effect of different am-

monia sources on aceticlastic and hydrogenotrophic methanogens. Bioresource

Technology 250, 390-397 (M2). https://doi.org/10.1016/j.biortech.2017.11.081

Tian, H., Fotidis, I.A., Mancini, E., Angelidaki, I., (2017). Different cultivation

methods to acclimatise ammonia-tolerant methanogenic consortia. Bioresource

Technology 232, 1-9 (M1). https://doi.org/10.1016/j.biortech.2017.02.034

Fotidis, I.A., Treu, L., Angelidaki, I. (2017). Enriched ammonia-tolerant meth-

anogenic cultures as bioaugmentation inocula in continuous biomethanation pro-

cesses. Journal of Cleaner Production 166, 1305-1313 (M1-M3).

https://doi.org/10.1016/j.jclepro.2017.08.151Get rights and content

Mahdy, A., Fotidis, I.A., Mancini, E., Ballesteros, M., González-Fernández, C.,

Angelidaki, I. (2017). Ammonia tolerant inocula provide a good base for anaer-

obic digestion of microalgae in 3rd generation biogas process. Bioresource Tech-

nology 225: 272-278 (M1-M3). https://doi.org/10.1016/j.biortech.2016.11.086










https://doi.org/10.1016/j.jclepro.2017.08.151

https://doi.org/10.1016/j.jclepro.2017.08.151


Wang, H., Fotidis, I.A., and Angelidaki, I. (2016). Ammonia-LCFA synergetic co-

inhibition effect in manure-based continuous biomethanation process. Biore-

source Technology 209: 282-289 (M2).


Wang, H., Fotidis, I.A., Angelidaki, I. (2015). Ammonia effect on hydrogen-

otrophic methanogens and syntrophic acetate oxidizing bacteria. FEMS Microbi-

ology Ecology 91 (M2). https://doi.org/10.1093/femsec/fiv130

1.6.2 ISI papers under preparation

Fotidis, I.A., Moretti, S., Damgaard, A., Astrup, T., Angelidaki, I. Environmental

and cost assessment of bioaugmentation to alleviate ammonia inhibition on bio-

methanation processes (M4).

Yan, M., Fotidis, I.A., Jéglot, A., Treu, L., Tian, H., Palomoa, A., Zhu, X., An-

gelidaki, I., Long term preservation and viability of ammonia tolerant methano-

genic cultures (M1-M3).

Yan, M., Campanaro, S., Treu, L., Tian, H., Zhu, X., Angelidaki, I., Fotidis, I.A.

Insights into ammonia adaptations and methanogenic precursor oxidation by ge-

nome-guided analysis (M1-M3)

Yan, M., Campanaro, S., Treu, L., Tian, H., Khoshnevisan, B., Tsapekos, P., An-

gelidaki, I., Fotidis, I.A. Bioaugmentation contributes to stable anaerobic diges-

tion of organic fraction of municipal solid waste under extreme ammonia levels

(M3).

Yan, M., Jéglot, A., Angelidaki, I., Fotidis, I.A. Novel cultivation strategies of am-

monia tolerant methanogenic consortia (M1).

Yan, M., Caetano, M.I.S.L., Angelidaki, I., Fotidis, I.A. The critical biomass for

successful bioaugmentation in ammonia-inhibited anaerobic reactors (M3).

1.6.3 Conferences, oral presentations and posters

Ioannis Fotidis, Hailin Tian, Enrico Mancini, Laura Treu, Irini Angelidaki. Bioaug-

mentation alleviates ammonia inhibition during protein-rich substrate bio-

methanation. 16th World Congress of Anaerobic Digestion, Delft, June 23-27,

2019, poster, (M3).

Miao Yan, Hailin Tian, Benyamin Khoshnevisan, Panagiotis Tsapekos, Irini An-

gelidaki, Ioannis Fotidis, Continuous biomethanation of organic fraction of mu-

nicipal solid waste under extreme ammonia levels. 16th World Congress of An-

aerobic Digestion, Delft, June 23-27, 2019, conference paper (M3).

Miao Yan, Hailin Tian, Ioannis Fotidis, Benyamin Khoshnevisan, Panagiotis Tsa-

pekos, Irini Angelidaki, Ammonia inhibition threshold during continuous bio-

methanation process. Sustain DTU conference-Creating Technology for a Sus-

tainable Society, Kgs. Lyngby, Denmark, December 29-30, 2018, Abstract,

poster and short oral presentation (M2).

Hailin Tian, Ioannis Fotidis, Laura Treu, Ahmed Mahdy, Mercedes Ballesteros,

Cristina González-Fernández, Irini Angelidaki,. Acclimation to extremely high

ammonia levels during continuous biomethanation process. 15th World Congress

of Anaerobic Digestion, Beijing, October 17-20, 2017, conference paper and oral

presentation (M1).

Hailin Tian, Ioannis Fotidis, Enrico Mancini, Irini Angelidaki. Combined inhibitory

effect of ammonia and LCFA on biomethanation process. 15th World Congress of

Anaerobic Digestion, Beijing, October 17-20, 2017, conference paper and oral

presentation (M2).



https://doi.org/10.1093/femsec/fiv130

Ioannis Fotidis, Hailin Tian, Enrico Mancini, Irini Angelidaki. Batch, fed-batch and

CSTR reactors as cultivation systems to acclimate ammonia tolerant methano-

gens. 15th World Congress of Anaerobic Digestion, Beijing, October 17-20, 2017,

poster (M1).

Hailin Tian, Ioannis A. Fotidis, Enrico Mancini, Irini Angelidaki. Microbial commu-

nity dynamics during a successful acclimation process to extremely high ammo-

nia levels in continuous anaerobic digester. Sustain DTU conference-Creating

Technology for a Sustainable Society, Kgs. Lyngby, Denmark, December 6, 2017,

abstract, poster and short oral presentation (M1, M2).

Enrico Mancini, Hailin Tian, Ioannis A. Fotidis, Irini Angelidaki. Acclimation of

ammonia tolerant methanogenic consortia using different bioreactor types. Sus-

tain DTU conference-Creating Technology for a Sustainable Society, Kgs. Lyngby,

Denmark, December 6, 2017, abstract, poster and short oral presentation (M1).

Hailin Tian, Enrico Mancini, Ioannis Fotidis, Irini Angelidaki. Acclimation of con-

tinuous biomethanation process to extremely high ammonia levels. Biogas Sci-

ence, Szeged, Hungary, 21-24 August 2016, abstract and oral presentation (M1).

Ioannis Fotidis, Han Wan, Irini Angelidaki. Bioaugmentation of ammonia tolerant

enriched methanogenic cultures: A microbiological process to efficiently digest

ammonia-rich biomasses. 4th International Conference on Sustainable Solid

Waste Management, Limassol, Cyprus June 23–25, 2016, abstract and oral

presentation (M2, M3).

Han Wan, Ioannis Fotidis, Irini Angelidaki. Effect of ammonia on hydrogen-

otrophic methanogens and syntrophic acetate oxidizing bacteria. Sustain DTU

conference-Creating Technology for a Sustainable Society, Kgs. Lyngby, Den-

mark, December 17, 2015, abstract, poster and short oral presentation (M2).

Ioannis Fotidis, Han Wan, Irini Angelidaki. Ammonia tolerant enriched methano-

genic cultures as bioaugmentation inocula to alleviate ammonia inhibition in con-

tinuous anaerobic reactors. 14th World Congress of Anaerobic Digestion, Viña del

Mar, Chile, November 15-18, 2015, conference paper, poster and oral presenta-

tion (M2, M3).

1.6.4 Utilization of project results

We believe that the results obtained in this project provide a good base for exploring

the potential full scale application of the developed bioaugmentation technology. As

it was mentioned before, the current project was focusing on technology development

and pilot-scale application and not on commercialization of the process. Thus, there

was no obligation for business plan development. With this in mind, we believe that

the developed technology could be utilized commercially in the near future by the

biogas plant operators due to the significant economic and environmental benefits

that offers (based on the systems analysis 1.5.4).

Characteristically, animal manures (especially cow and pig) are a fundamental ele-

ment for a healthy high rate methane production. However, manures have low me-

thane yields and thus is necessary to co-digest them with other carbon-rich bio-

masses (up to 25% of co-substrate in Denmark), to increase productivity and render

the process economical profitable. The amounts of the available co-substrates are

limited and the need for new co-substrates with high methane yields growing expo-

nentially the last years as the Danish government has set the goal of 50% use of all

animal slurries in anaerobic reactors. This goal is very optimistic since the current

use of animal slurries is estimated to be less than 20%.

MicrobStopNH3 project developed and tested an innovative microbiological process

that alleviates the ammonia inhibition effect in anaerobic reactors and thus allows


the digestion of ammonia-rich wastes. These unique characteristics of MicrobStopNH3

justify the necessity for a future, full-scale application of the developed technology.

Since ammonia-rich substrates (e.g. food waste, slaughterhouse waste, mink and

poultry manures, etc.) are abundant in Denmark and worldwide but, even if they

have high methane potentials, are not widely used by the biogas plants today since

they pose a thread for the stability and the efficiency of methane production. Addi-

tionally, it has been proven that many biogas plants are suffering from ammonia

induced “inhibited steady-state” despite the fact that avoid using large amounts of

ammonia-rich substrates. The conventional methods to tackle the ammonia toxicity

problem (dilution, temperature lowering, etc.) are expensive, time consuming, re-

duce the reactor performance and don’t provide a long-term solution to the problem.

Thus, the potential use of the MicrobStopNH3 bioaugmentation technology will allow

these abundant but unwanted ammonia-rich biomasses to be used as co-substrates

in the manure-based biogas reactors, enhancing their productivity and consequently

their profitability.

We strongly believe that the MicrobStopNH3 technology could counteract the ammo-

nia toxicity effect on the anaerobic reactors efficiency, and thus ammonia-rich sub-

strates could cover a large part of the need for additional co-digestion substrates that

the Danish clean energy policies require. Even though the MicrobStopNH3 project has

shown great potentials for the bioaugmentation process by fulfilling all its objectives;

there is still a lack of full-scale process approach and design for the commercial ex-

ploitation of the technology. This will have implications for the energy policy and

future energy supply system in Denmark as well as in other countries. MicrobStopNH3

technology can further increase the interest for investment in the biogas sector, can

promote more construction activities in the biogas sector to fulfil the ambition of

100% fossil-free energy supply and reduced greenhouse gas emissions from 80% to

95%, by year 2050. The improved process efficiency and plant economy can also

contribute to reduce the direct and indirect governmental subsidies. While, a suc-

cessful biogas industry will allow Denmark to be less dependent on the natural gas

imported from external, “unpredictable” suppliers.

As it has presented in section 1.5 (Project results and dissemination), three PhD

students have participated in the project activities and many of the results of the

project were part of their PhD thesis and the ISI papers that they have produced.

During the project, some experimental activities were used during the 12136 Bioen-

ergy technologies course (DTU) to train MSc and PhD students in ammonia inhibited

anaerobic digestion in lab-scale activities. It is estimated that more than 40 students

have been part of these activities between 2015 and 2019. Finally, at this stage the

MicrobStopNH3 partners do not consider applying for any patents.


https://kurser.dtu.dk/course/12136

https://kurser.dtu.dk/course/12136

1.7 Project conclusion and perspective

The MicrobStopNH3 project proposed bioaugmentation as a novel technical approach

to establish ammonia tolerant consortia in continuous digesters operating under high

ammonia levels. The major conclusion of the project is that: it is technically possible

to use bioaugmentation of ammonia tolerant methanogenic consortia to improve sig-

nificantly (up to 40%) methane production of continuous anaerobic reactors suffering

from ammonia toxicity.

During the project many lab-scale experiments and assessments were performed,

thus there are many significant scientific conclusions drawn from them that have or

will be included in the ISI publications of the project. With that in mind the, the main

conclusions of the project are:

Using the case-appropriate acclimatization strategy it is possible to create am-

monia tolerant methanogenic consortia in batch, fed-batch and continuous reac-

tors.

Mixed mesophilic and thermophilic ammonia tolerant methanogenic consortia

can be used as bioaugmentation inocula with similar or better efficiency com-

pared to pure methanogenic cultures.

The “critical biomass” (minimum amount) of bioaugmentation inoculum neces-

sary for a successful bioaugmentation in continuous reactors is at least 2% v/v.

The hydrogenotrophic methanogens are the keystone microbes for the success

of the bioaugmentation process under extreme ammonia conditions, since they

immediately reduce the hydrogen partial pressure, which affects positively the

efficiency of many other catabolic processes.

The pilot-scale verification of the developed bioaugmentation technology showed

that the improvement in methane production is similar to the one achieved in

the lab-scale continuous reactors' experiments.

The developed bioaugmentation technology, if applied in full scale anaerobic re-

actors, could have a clearly positive effect on the climate change and emissions

avoidance as well as a significant economic benefits for the biogas plants

We strongly believe that the technology developed in the MicrobStopNH3 project can

be utilised in full-scale reactors. In order to achieve this, the technology readiness

level has to be moved pass the pilot-scale verification stage, the stakeholders in

collaboration with the researchers have to invest funds and time in order to create a

commercially applicable process that meets the requirements and the expectations

of the industry. This commercially available bioaugmentation technology is the next

major goal of the researchers involved in the MicrobStopNH3 project as well as other

researchers around the world that they have recently included bioaugmentation in

their research activities, inspired by the MicrobStopNH3 project results.


Annex

The first pages of the 12 Published ISI papers


Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier.com/locate/biortech

Hydrogenotrophic methanogens are the key for a successfulbioaugmentation to alleviate ammonia inhibition in thermophilic anaerobicdigesters

Hailin Tiana,b, Miao Yana, Laura Treua,c, Irini Angelidakia, Ioannis A. Fotidisa,⁎

a Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, 2800 Kgs. Lyngby, Denmarkb Environmental Research Institute, National University of Singapore, 1 Create Way, Create Tower, Singapore 138602, Singaporec Department of Biology, University of Padua, 35131 Padua, Italy

G R A P H I C A L A B S T R A C T

A R T I C L E I N F O

Keywords:BiogasAmmonia-tolerant consortiumMicrobial communityMethanoculleus thermophilusMethanosarcina thermophila

A B S T R A C T

Bioaugmentation to alleviate ammonia inhibition under thermophilic anaerobic digestion has never been re-ported, as well as the working mechanism that allows a fast and successful bioaugmentation. Thus twobioaugmentation inocula (an enriched culture, and a mixed culture composed 50/50 by Methanoculleus ther-mophilus and the enriched culture) on the recovery of ammonia-inhibited thermophilic continuous reactors wasassessed. The results showed that bioaugmentation improved methane yield by 11–13% and decreased thevolatile fatty acids (VFA) by 45–52% compared to the control reactor (abiotic augmentation). Moreover, theimportance of hydrogenotrophic methanogens to a fast and successful bioaugmentation was recognized.Specifically, the instant hydrogen partial pressure reduction by the bioaugmented hydrogenotroph createdthermodynamically favourable conditions for the acetate oxidation process and consequently, the catabolism ofother VFA. High-throughput sequencing results strengthened this explanation by showing that the bioaugmentedM. thermophilus stimulated the growth of syntrophic acetate oxidising bacterium Thermacetogenium phaeum,immediately after bioaugmentation.

https://doi.org/10.1016/j.biortech.2019.122070Received 25 May 2019; Received in revised form 20 August 2019; Accepted 24 August 2019

⁎ Corresponding author. Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, DK-2800 Kgs. Lyngby,Denmark.

E-mail address: [email protected] (I.A. Fotidis).

Bioresource Technology 293 (2019) 122070

Available online 27 August 20190960-8524/ © 2019 Elsevier Ltd. All rights reserved.

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Chemosphere 231 (2019) 415e422

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Chemosphere

journal homepage: www.elsevier .com/locate/chemosphere

Bioaugmentation strategy for overcoming ammonia inhibition duringbiomethanation of a protein-rich substrate

Hailin Tian, Enrico Mancini, Laura Treu, Irini Angelidaki, Ioannis A. Fotidis*

Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, DK-2800 Kgs. Lyngby, DK

h i g h l i g h t s

* Corresponding author. Department of EnvironmUniversity of Denmark, Bygningstorvet BygningDenmark.


https://doi.org/10.1016/j.chemosphere.2019.05.1400045-6535/© 2019 Elsevier Ltd. All rights reserved.

g r a p h i c a l a b s t r a c t

� Bioaugmentation alleviated ammoniainhibition of a reactor fed withmicroalgae.

� Bioaugmented M. bourgensisincreased methane yield by 28% anddecreased VFA by 80%.

� Reduction of hydrogen pressure andpropionate is crucial in alleviatinginhibition.

� The bioaugmentation success lies onthe triggered “microbiological dom-ino effect”.

� M. soligelidi was the dominantmethanogen three HRTs afterbioaugmentation.

a r t i c l e i n f o

Article history:Received 20 December 2018Received in revised form16 May 2019Accepted 17 May 2019Available online 22 May 2019

Handling Editor: Y Liu

Keywords:MethaneAmmonia inhibitionMicrobial communityMethanoculleus bourgensisHydrogen partial pressure

a b s t r a c t

High ammonia levels inhibit anaerobic digestion (AD) process and bioaugmentation with ammoniatolerant methanogenic culture is proposed to alleviate ammonia inhibition. In the current study,hydrogenotrophic Methanoculleus bourgensis was bioaugmented in an ammonia-inhibited continuousreactor fed mainly with microalgae (a protein-rich biomass), at extreme ammonia levels (i.e. 11 g NH4

þ-NL�1). The results showed 28% increase in methane production immediately after bioaugmentation.Moreover, volatile fatty acids decreased rapidly from more than 5 g L�1 to around 1 g L�1, with a fastreduction in propionate concentration. High throughput 16s rRNA gene sequencing demonstrated thatthe bioaugmented M. bourgensis doubled its relative abundance after bioaugmentation. “Microbiologicaldomino effect”, triggered by the bioaugmented M. bourgensis establishing a newly efficient community,was proposed as the working mechanism of the successful bioaugmentation. Additionally, a strongaceticlastic methanogenesis was found at the end of the experiment evidenced by the dominant pres-ence of Methanosarcina soligelidi and the low abundance of syntrophic acetate oxidising bacteria at thefinal period. Overall, for the first time, this study proved the positive effect of bioaugmentation onammonia inhibition alleviation of the microalgae-dominating fed reactor, paving the way of efficientutilization of other protein-rich substrates in the future.

© 2019 Elsevier Ltd. All rights reserved.

ental Engineering, Technical115, DK-2800 Kgs. Lyngby,

MicrobStopNH3-Final

1. Introduction

Biogas from anaerobic digestion (AD) is a renewable energycarrier, which plays an important role in substituting fossil fuel and

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Acclimatization contributes to stable anaerobic digestion of organic fractionof municipal solid waste under extreme ammonia levels: Focusing onmicrobial community dynamics

Miao Yana, Ioannis A. Fotidisa,⁎, Hailin Tiana, Benyamin Khoshnevisana, Laura Treub,Panagiotis Tsapekosa, Irini Angelidakia

a Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, DK-2800 Kgs. Lyngby, DenmarkbDepartment of Biology, University of Padova, Via U. Bassi 58/b, 35121 Padova, Italy


Keywords:Ammonia acclimatizationHydrogenotrophic pathwayAceticlastic pathwayMetabolic pathway shiftMethanosarcina soligelidi

A B S T R A C T

The organic fraction of municipal solid waste (OFMSW) is an abundant and sustainable substrate for theanaerobic digestion (AD) process, yet ammonia released during OFMSW hydrolysis could result in suboptimalbiogas production. Acclimatized ammonia tolerant microorganisms offer an efficient way to alleviate ammoniainhibition during AD. This study aimed to achieve an efficient AD of OFMSW under extreme ammonia levels andelucidate the dynamics of the acclimatized microbial community. Thus, two mesophilic continuous stirred tankreactors (CSTR), fed only with OFMSW, were successfully acclimatized up to 8.5 g NH4

+-N/L, and their methaneyields fluctuated< 10%, compared to the methane yields without ammonia addition. Microbiological analysesshowed that Methanosaeta concilii and Methanosarcina soligelidi were the dominant methanogens at low and highammonia levels, respectively. Whilst, a unique metabolic pathway shift, from aceticlastic to hydrogenotrophicmethanogenesis, of M. soligelidi was identified during the acclimatization process.

1. Introduction

The increasing global need for natural resources and consumergoods leads to vast amounts of municipal solid waste (MSW). In 2018,more than 2.5 Mt of MSW were generated in the European Union(Eurostat, 2018), with 46% of them been organic (OFMSW: organicfraction of MSW) (Hoornweg and Bhada-Tata, 2012). Improper treat-ment methods of OFMSW can cause environmental and health issues(Fisher, 2006). Currently, most of the MSW is treated in one of the fourways: landfilling (24%), incineration (28%), recycling (29%), andcomposting (16%) (Eurostat, 2018). Landfilling and incinerationmethods do not take advantage of the organic faction and the nutrientsof OFMSW and can lead to extra greenhouse gas emissions (Fisher,2006; Eurostat, 2018). At the same time, huge consumption andshortage of fossil fuels motivate researchers to find alternative energysources. Thus, new treatment technologies must be developed to notonly offset the use of fossil fuels but also maximize the reuse of the vastamounts of the OFMSW and thereby maintain and recycle the usefulnutrient back to agriculture.

Anaerobic digestion (AD) is a process that can convert organic wasteinto sustainable energy (biogas), via a series of interrelated microbial

metabolisms (Campanaro et al., 2018). The AD process is divided infour steps (i.e. hydrolysis, acidogenesis, acetogenesis and methano-genesis), which are mediated primarily by bacteria and archaea(Angelidaki et al., 2011). In addition, the liquid product of the ADprocess (digestate) contains high levels of nitrogen and phosphorus,which can be potentially reused as biofertilizer or after extraction, assupplement for fermentation processes. In spite of these benefits, thedegradation of OFMSW produces ammonia, a by-product from thecatabolism of proteins, which can be toxic to the AD process, resultingin poor operational stability and reduced methane production effi-ciency (Tian et al., 2018b).

Total ammonia (TAN) is the sum of ammonium ions (NH4+) and

free ammonia (FAN, NH3), while FAN exists in an equilibrium definedby the pH and the temperature (Tian et al., 2017). As Koster andLettinga (1988) reported, when ammonia concentrations ranged from 4to 5.7 g NH4

+-N/L, more than 56% of the methanogenic activity wasinhibited in a granular sludge reactor. The reason is that FAN is freelymembrane-permeable and hence, decreases methanogenic activity byinterfering with the natural intracellular biological pathways of themethanogens (e.g. inhibiting specific methane synthesizing enzymereaction) (Sprott and Patel, 1986). It is generally accepted that

https://doi.org/10.1016/j.biortech.2019.121376Received 22 March 2019; Received in revised form 19 April 2019; Accepted 20 April 2019

⁎ Corresponding author.E-mail address: [email protected] (I.A. Fotidis).

Bioresource Technology 286 (2019) 121376

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16s rRNA gene sequencing and radioisotopic analysis reveal the compositionof ammonia acclimatized methanogenic consortiaHailin Tian, Laura Treu, Konstantinos Konstantopoulos, Ioannis A. Fotidis⁎, Irini AngelidakiDepartment of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, DK-2800 Kgs. Lyngby, Denmark



Keywords:Anaerobic digestionEnriched cultureMethanogenic pathwayAmmonia-tolerant microbial communityMethanomassiliicoccus luminyensis

A B S T R A C T

Different mesophilic and thermophilic methanogenic consortia were acclimatised and enriched to extreme totalammonia (9.0 and 5.0 g NH4

+-N L−1, respectively) and free ammonia (1.0 and 1.4 g NH3-N L−1, respectively)levels in this study. [2-14C] acetate radioisotopic analyses showed the dominance of aceticlastic methanogenesisin all enriched consortia. According to 16S rRNA gene sequencing result, in mesophilic consortia, methylo-trophic Methanomassiliicoccus luminyensis was predominant, followed by aceticlastic Methanosarcina soligelidi. Apossible scenario explaining the dominance of M. luminyensis includes the use of methylamine produced byTissierella spp. and biomass build-up by metabolizing acetate. Nevertheless, further studies are needed to pin-point the exact metabolic pathway of M. luminyensis. In thermophilic consortia, aceticlastic Methanosarcinathermophila was the sole dominant methanogen. Overall, results derived from this study demonstrated the ef-ficient biomethanation ability of these ammonia-tolerant methanogenic consortia, indicating a potential appli-cation of these consortia to solve ammonia toxicity problems in future full-scale reactors.

1. Introduction

Anaerobic digestion (AD) is a widely used biotechnological processto treat a variety of biowaste and recover renewable energy (CH4). It isa complex biological process that is mediated by different microbes

(bacteria and archaea) and consists of four steps, named hydrolysis,acidogenesis, acetogenesis and methanogenesis. Acetate is the mostimportant precursor of methane, with two main pathways involved inits catabolism: aceticlastic methanogenesis (Eq. (1)) and syntrophicacetate oxidation (SAO) coupled with hydrogenotrophic

https://doi.org/10.1016/j.biortech.2018.09.128Received 2 August 2018; Received in revised form 24 September 2018; Accepted 25 September 2018

⁎ Corresponding author at: Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, DK-2800 Kgs. Lyngby,Denmark.


Bioresource Technology 272 (2019) 54–62

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Effect of different ammonia sources on aceticlastic and hydrogenotrophicmethanogens

Hailin Tian, Ioannis A. Fotidis⁎, Konstantinos Kissas, Irini AngelidakiDepartment of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, DK-2800 Kgs. Lyngby, Denmark



Keywords:Ammonia inhibitionAmmonium chlorideAnaerobic digestionPure strainUrea

A B S T R A C T

Ammonium chloride (NH4Cl) was usually used as a model ammonia source to simulate ammonia inhibitionduring anaerobic digestion (AD) of nitrogen-rich feedstocks. However, ammonia in AD originates mainly fromdegradation of proteins, urea and nucleic acids, which is distinct from NH4Cl. Thus, in this study, the inhibitoryeffect of a “natural” ammonia source (urea) and NH4Cl, on four pure methanogenic strains (aceticlastic:Methanosarcina thermophila,Methanosarcina barkeri; hydrogenotrophic: Methanoculleus bourgensis,Methanoculleusthermophilus), was assessed under mesophilic (37 °C) and thermophilic (55 °C) conditions. The results showedthat urea hydrolysis increased pH significantly to unsuitable levels for methanogenic growth, while NH4Cl had anegligible effect on pH. After adjusting initial pH to 7 and 8, urea was significantly stronger inhibitor with longerlag phases to methanogenesis compared to NH4Cl. Overall, urea seems to be more toxic on both aceticlastic andhydrogenotrophic methanogens compared to NH4Cl under the same total and free ammonia levels.

1. Introduction

Biogas (a mixture of CH4 and CO2) is an attractive renewable energy(Holm-Nielsen et al., 2009), which is formed during anaerobic digestion(AD) of different biomasses. As one of the most promising and widelyused green technologies, AD is a complex biological process with dif-ferent microorganisms involved, which can reduce the waste pollution

and offset part of the energy usage (Chynoweth et al., 2001). However,it is reported that some potential substrates are toxic to AD process byinhibiting the microorganisms' activity (Chen et al., 2008). Amongthese substrates, nitrogen-rich substrates stand out, due to the ammoniaformation during their degradation. A low ammonia concentration(< 200mgNH4

+-N L−1) is beneficial to AD process; nevertheless, re-latively high ammonia levels (> 2000mgNH4

+-N L−1) would inhibit

https://doi.org/10.1016/j.biortech.2017.11.081Received 6 November 2017; Received in revised form 22 November 2017; Accepted 23 November 2017



Available online 24 November 20170960-8524/ © 2017 Elsevier Ltd. All rights reserved.

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Acclimation to extremely high ammonia levels in continuousbiomethanation process and the associated microbial community dynamics

Hailin Tiana, Ioannis A. Fotidisa,⁎, Enrico Mancinia, Laura Treua,b, Ahmed Mahdyc,Mercedes Ballesterosd,e, Cristina González-Fernándezd, Irini Angelidakia

a Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, DK-2800 Kgs. Lyngby, Denmarkb Department of Agronomy, Food, Natural Resources, Animal and Environment (DAFNAE), Viale dell’Università, 16, 35020 Legnaro, Padova, Italyc Department of Agricultural Microbiology, Faculty of Agriculture, Zagazig University, 44511 Zagazig, Egyptd Biotechnological Processes for Energy Production Unit – IMDEA Energy, 28935 Móstoles, Madrid, Spaine Biofuels Unit – Research Center for Energy, Environment and Technology (CIEMAT), 28040 Madrid, Spain



Keywords:MethaneAmmonia inhibitionMicrobial communitySyntrophic acetate oxidizerMethanosarcina

A B S T R A C T

Acclimatized anaerobic communities to high ammonia levels can offer a solution to the ammonia toxicityproblem in biogas reactors. In the current study, a stepwise acclimation strategy up to 10 g NH4

+-N L−1, wasperformed in mesophilic (37 ± 1 °C) continuously stirred tank reactors. The reactors were co-digesting (20/80based on volatile solid) cattle slurry and microalgae, a protein-rich, 3rd generation biomass. Throughout theacclimation period, methane production was stable with more than 95% of the uninhibited yield. Next gen-eration 16S rRNA gene sequencing revealed a dramatic microbiome change throughout the ammonia acclima-tion process. Clostridium ultunense, a syntrophic acetate oxidizing bacteria, increased significantly alongside withhydrogenotrophic methanogen Methanoculleus spp., indicating strong hydrogenotrophic methanogenic activityat extreme ammonia levels (> 7 g NH4

+-N L−1). Overall, this study demonstrated for the first time that ac-climation of methanogenic communities to extreme ammonia levels in continuous AD process is possible, bydeveloping a specialised acclimation AD microbiome.

1. Introduction

Anaerobic digestion (AD) is a sustainable technology that can pro-duce biogas and nutrient-rich bio-fertilizer from a broad variety of re-sidual biomass (e.g. agricultural waste, food waste, and sewage sludge)(Karim et al., 2005). AD is a complex biological process, which

comprises four main steps: hydrolysis, acidogenesis, acetogenesis andmethanogenesis, with a variety of microorganisms mediating each step.Acetate is the main precursor of methane production which follows twomajor methanogenic pathways: a) aceticlastic pathway and b) hydro-genotrophic pathway (syntrophic acetate oxidation (SAO) coupled withhydrogenotrophic methanogenesis). Aceticlastic pathway is mediated

http://dx.doi.org/10.1016/j.biortech.2017.09.148Received 21 August 2017; Received in revised form 19 September 2017; Accepted 21 September 2017



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Chemical Engineering Journal

journal homepage: www.elsevier.com/locate/cej

A proposed mechanism for the ammonia-LCFA synergetic co-inhibitioneffect on anaerobic digestion process

Hailin Tian, Panagiotis Karachalios, Irini Angelidaki, Ioannis A. Fotidis⁎

Department of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, DK-2800 Kgs. Lyngby, Denmark

H I G H L I G H T S

• Identification of NH3-LCFA synergeticeffect in batch and continuous re-actors.

• A new mechanism was proposed toexplain the NH3-LCFA synergetic ef-fect.

• Excess LCFA levels due to β-oxidationinhibition by NH3 trigger the syner-gism.

• Adaptation of the microbiome to thesynergism is possible in continuousreactor.

• Different NH3-LCFA levels cause thesynergism in batch and continuousreactors.



Keywords:Anaerobic microbiomeBatch reactorsCSTR reactorsMethaneToxicity

A B S T R A C T

Ammonia and long chain fatty acids (LCFA) are two major inhibitors of the anaerobic digestion (AD) process.The individual inhibitory effect of each of these two inhibitors is well established; however, the combined co-inhibition effect has not been thoroughly assessed yet. In the current study, the ammonia-LCFA synergetic co-inhibition effect was investigated in both batch and continuous experiments. In the batch experiments, a clearammonia-LCFA synergetic co-inhibitory effect was identified when the LCFA concentrations were higher than0.05 g oleate L−1 and ammonia levels between 4.0 and 7.0 NH4

+-N L−1. This synergetic effect for LCFA andammonia levels above 1.1 g oleate L−1 and 4.5 NH4

+-N L−1, respectively, was validated in continuous reactorsexperiments. Nevertheless, adaptation of the AD microbiome to this synergetic co-inhibition could occur after aperiod of continuous operation. A potential mechanism to explain the synergetic co-inhibition lies on the initialinhibition of methanogens caused by ammonia resulting in increased VFA and hydrogen concentrations, whichin turn renders β-oxidation of LCFA thermodynamically unfavourable and thereby brings about further excessaccumulation of LCFA and consequently higher unspecific toxicity of all AD steps. This is a vicious cycle, whichmakes the combined inhibition of the two toxicants more severe, compared to the sum of their individualinhibition effects at the same operational conditions.

1. Introduction

Anaerobic digestion (AD) is a widely used sustainable technologyfor bioenergy (CH4) recovery from a variety of biowastes and

wastewaters, such as industrial wastewater, agricultural and forestryresidues, municipal sewage sludge etc. [1,2]. It is a complex biologicalprocess, consisting of four steps, i.e. hydrolysis, acidogenesis, acet-ogenesis and methanogenesis, which are mediated by different groups

https://doi.org/10.1016/j.cej.2018.05.083Received 6 March 2018; Received in revised form 9 May 2018; Accepted 13 May 2018


Chemical Engineering Journal 349 (2018) 574–580

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Journal of Cleaner Production 166 (2017) 1305e1313

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Journal of Cleaner Production

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Enriched ammonia-tolerant methanogenic cultures asbioaugmentation inocula in continuous biomethanation processes

Ioannis A. Fotidis*, Laura Treu, Irini AngelidakiDepartment of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, DK-2800, Kgs. Lyngby, Denmark


Article history:Received 1 June 2017Received in revised form17 August 2017Accepted 17 August 2017Available online 19 August 2017

Keywords:Ammonia toxicityArchaeonAceticlastic methanogenBacteriumHydrogenotrophic methanogen

* Corresponding author.E-mail address: [email protected] (I.A. Fotidis).

http://dx.doi.org/10.1016/j.jclepro.2017.08.1510959-6526/© 2017 Elsevier Ltd. All rights reserved.

a b s t r a c t

Many ammonia-rich biomass sources, such as manures and protein-rich substrates, are potential in-hibitors of the anaerobic digestion (AD) process. It was previously demonstrated that bioaugmentation ofMethanoculleus bourgensis MS2T in an ammonia inhibited process in a continuous stirred tank reactor(CSTR), resulted in up to 90% recovery of the methane production compared to the uninhibited pro-duction. However, cultivation of pure strains has practical difficulties due to the need of special growthmedia and sterile conditions. In contrast, acclimatized enriched cultures have minor sterility re-quirements. In the current study, an enriched ammonia-tolerant methanogenic culture was bio-augmented in a CSTR reactor operating under ammonia-induced, inhibited-steady-state. The resultsdemonstrated that bioaugmentation, completely counteracted the ammonia toxicity effect. This indicatesthat a commercial application of bioaugmentation could improve up to 36% the methane production, thegreenhouse gas reduction efficiency and the gross revenue of ammonia inhibited full scale biogas re-actors. 16S rRNA gene sequencing showed that bioaugmentation changed the microbial composition ofthe reactors resulting in higher bacterial and lower archaeal community diversity. The bioaugmentedreactor showed a fourfold increase of the abundance of the bioaugmented methanogens compared to thecontrol reactor. This indicates that ammonia-tolerant methanogens established well in the ammonia-inhibited reactor and dominated over the domestic methanogenic population. Finally, this studyshowed that the enriched culture alleviated ammonia toxicity 25% more efficiently than the previouslyused pure culture.

© 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Vast amounts of ammonia-rich organic wastes are producedyearly from the agricultural and the food industrial sectors (Kov�acset al., 2013). Anaerobic digestion (AD) is one of the most effectivemethods to treat this waste, as it provides energy (methane) and abio-fertilizer (digestate) (Tampio et al., 2016). Moreover, somemanures (e.g. pig, poultry etc.) that are often used as substrates inbiogas reactors contain high amounts of urea. Ammonia is a well-known inhibitor of the AD process (Westerholm et al., 2015). Ithas been widely shown that free ammonia (unionised, NH3) is themost toxic form of the total ammonia (NH4

þþNH3) for the meth-anogenic communities mediating the AD process (Rajagopal et al.,2013). The NH3 levels depend on the total ammonia concentra-tion in a reactor and on the NH4

þ/NH3 equilibrium, which is affected

MicrobStopNH3-Final

by the temperature and the pH (Yenigün and Demirel, 2013).Many solutions have been proposed to solve the ammonia in-

hibition problem. Up to now, the two most common methods arelowering the operating temperature and diluting the reactor con-tent with water (Kelleher et al., 2002; Nielsen and Angelidaki,2008). Nevertheless, these methods can counteract the ammoniatoxicity only to a limited extend, are uneconomical and do notprovide a permanent solution (Mass�e et al., 2014).

Europe has currently more than 17,240 biogas plants and mostof them use combined heat and power (CHP) units to generateelectricity on site, with an average efficiency of 40% (EuropeanBiogas Association, 2015; Herbes et al., 2016). At the same time, ithas been found that several of the European biogas reactors areseriously affected by ammonia toxicity, leaving unexploited morethan 30% of their methane potential, while operating in anammonia induced inhibited-steady-state (Duan et al., 2012; Fotidiset al., 2014a). This suboptimal, but apparently stable process isresulting in severe operational problems with increased CO2

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Different cultivation methods to acclimatise ammonia-tolerantmethanogenic consortia

http://dx.doi.org/10.1016/j.biortech.2017.02.0340960-8524/� 2017 Elsevier Ltd. All rights reserved.

⇑ Corresponding author.E-mail address: [email protected] (I.A. Fotidis).


Hailin Tian, Ioannis A. Fotidis ⇑, Enrico Mancini, Irini AngelidakiDepartment of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, DK-2800 Kgs. Lyngby, Denmark

h i g h l i g h t s

� Fed-batch was the most efficientmethod to acclimatise ammoniatolerant consortia.

� Fast acclimation of methanogens atextremely high FAN levels(1633 mg NH3-N L�1).

� Hydrogenotrophic methanogens weredominant at FAN levels above540 mg NH3-N L�1.

� CSTR acclimation failed at low TANlevel (<4.6 g NH4

+-N L�1) due towashout effect.

� Fed-batch is a promising acclimationmethod to be coupled withbioaugmentation.



Article history:Received 24 December 2016Received in revised form 7 February 2017Accepted 8 February 2017Available online 11 February 2017

Keywords:Batch reactorFed-batch reactorCSTRSpecific methanogenic activityIncubation time

a b s t r a c t

Bioaugmentation with ammonia tolerant-methanogenic consortia was proposed as a solution to over-come ammonia inhibition during anaerobic digestion process recently. However, appropriate technologyto generate ammonia tolerant methanogenic consortia is still lacking. In this study, three basic reactors(i.e. batch, fed-batch and continuous stirred-tank reactors (CSTR)) operated at mesophilic (37 �C) andthermophilic (55 �C) conditions were assessed, based on methane production efficiency, incubation time,TAN/FAN (total ammonium nitrogen/free ammonia nitrogen) levels and maximum methanogenic activ-ity. Overall, fed-batch cultivation was clearly the most efficient method compared to batch and CSTR.Specifically, by saving incubation time up to 150%, fed-batch reactors were acclimatised to nearly 2-fold higher FAN levels with a 37%–153% methanogenic activity improvement, compared to batch method.Meanwhile, CSTR reactors were inhibited at lower ammonia levels. Finally, specific methanogenic activitytest showed that hydrogenotrophic methanogens were more active than aceticlastic methanogens in allFAN levels above 540 mg NH3-N L�1.

� 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Anaerobic digestion (AD) is one of the most commonly usedmethods to treat a vast array of organic waste-slurries andwastewaters derived from different sources (e.g. agricultural

waste, industrial waste, food waste, municipal sewage sludgeetc.), which result in energy recovery (biogas; a mixture of CH4

and CO2) and in a nutrient-rich digestate used as biofertilizer(Bekkering et al., 2015). Additionally, AD reduces the greenhousegas emissions and has lower energy requirements compared toother waste treatment methods (Westerholm et al., 2012). How-ever, when ammonia-rich waste (e.g. animal manure, slaughter-house wastewater etc.) are used as AD substrates, an instability






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Ammonia tolerant inocula provide a good base for anaerobic digestion ofmicroalgae in third generation biogas process


⇑ Corresponding author.E-mail address: [email protected] (I.A. Fotidis).


Ahmed Mahdy a,b, Ioannis A. Fotidis c,⇑, Enrico Mancini c, Mercedes Ballesteros a,d,Cristina González-Fernández a, Irini Angelidaki c

aBiotechnological Processes for Energy Production Unit – IMDEA Energy, 28935 Móstoles, Madrid, SpainbDepartment of Agricultural Microbiology, Faculty of Agriculture, Zagazig University, 44511 Zagazig, EgyptcDepartment of Environmental Engineering, Technical University of Denmark, Bygningstorvet Bygning 115, DK-2800 Kgs. Lyngby, DenmarkdBiofuels Unit – Research Center for Energy, Environment and Technology (CIEMAT), 28040 Madrid, Spain

h i g h l i g h t s

� Efficient 3rd generation biogasproduction from protein-richmicroalgae.

� Synergistic co-digestion occurredonly when ‘‘algae VS”/‘‘cattle manureVS” >1.

� High methane yield for algaeachieved when ammonia tolerantinoculum was used.

� The highest yield was reached bycoupling codigestion and ammoniatolerant inoculum.



Article history:Received 14 October 2016Received in revised form 20 November 2016Accepted 21 November 2016Available online 23 November 2016

Keywords:Ammonia inhibitionAnaerobic digestionBMPCo-digestionMicroalgae

a b s t r a c t

This study investigated the ability of an ammonia-acclimatized inoculum to digest efficiently protein-richmicroalgae for continuous 3rd generation biogas production. Moreover, we investigated whetherincreased C/N ratio could alleviate ammonia toxicity. The biochemical methane potential (BMP) of fivedifferent algae (Chlorella vulgaris)/manure (cattle) mixtures showed that the mixture of 80/20 (on VSbasis) resulted in the highest BMP value (431 mL CH4 g VS�1), while the BMP of microalgae alone(100/0) was 415 mL CH4 g VS�1. Subsequently, anaerobic digestion of those two substrates was testedin continuous stirred tank reactors (CSTR). Despite of the high ammonium levels (3.7–4.2 g NH4

+-N L�1), CSTR reactors using ammonia tolerant inoculum resulted in relatively high methane yields (i.e.77.5% and 84% of the maximum expected, respectively). These results demonstrated that ammonia toler-ant inocula could be a promising approach to successfully digest protein-rich microalgae and achieve a3rd generation biogas production.


1. Introduction

A great effort has been dedicated to the development of greentechnologies for producing renewable biofuels. Conversion of

organic matter into biogas through anaerobic digestion (AD) hasbeen intensely investigated since, unlike bioethanol or biodiesel,it holds the possibility to convert a broad variety of organic sub-strate to biogas (Naik et al., 2010). However, deeper understandingis required to address several challenges that can affect perfor-mance and stability of the conversion process when using newfeedstocks. Microalgae, for example, seem to be a promising candi-











Ammonia–LCFA synergetic co-inhibition effect in manure-basedcontinuous biomethanation process


⇑ Corresponding author. Tel.: +45 45251418; fax: +45 45933850.E-mail address: [email protected] (I.A. Fotidis).


Han Wang, Ioannis A. Fotidis ⇑, Irini AngelidakiDepartment of Environmental Engineering, Technical University of Denmark, Building 113, DK-2800 Kgs. Lyngby, Denmark

h i g h l i g h t s

� An ‘‘ammonia–LCFA synergetic co-inhibition” effect was identified.

� With lipids as co-substrates it is notpossible to overcome ammoniatoxicity on AD.

� Co-digestion with carbohydrate wasmore robust to ammonia toxicitythan with lipid.

� Hydrogenotrophic archaea were morerobust to ammonia–LCFA synergeticco-inhibition.



Article history:Received 22 January 2016Received in revised form 26 February 2016Accepted 1 March 2016Available online 8 March 2016

Keywords:Ammonia toxicityAnaerobic digestionCo-digestionHydrogenotrophic methanogensLipids

a b s t r a c t

In the current study it has been hypothesized that, when organic loading of an anaerobic reactor isincreased, the additional cell biomass biosynthesis would capture more ammonia nitrogen and therebyreduce the ammonia toxicity. Therefore, the alleviation of the toxicity of high ammonia levels using lipids(glycerol trioleate-GTO) or carbohydrates (glucose-GLU) as co-substrates in manure-based thermophiliccontinuous stirred-tank reactors (RGTO and RGLU, respectively) was tested. At 5 g NH4

+-N L�1, relativemethane production of RGTO and RGLU, was 10.5% and 41% compared to the expected uninhibited produc-tion, respectively. At the same time control reactor (RCTL), only fed with manure, reached 32.7% comparedto the uninhibited basis production. Therefore, it seems that using lipids to counteract the ammoniaeffect in CSTR reactors creates an ‘‘ammonia–LCFA (long chain fatty acids) synergetic co-inhibition”effect. Moreover, co-digestion with glucose in RGLU was more robust to ammonia toxicity compared toRCTL.


1. Introduction

Anaerobic digestion (AD) process, is a sustainable technologythat is being used widely for organic waste treatment and concur-rent bioenergy (i.e. biogas) production (Rajagopal et al., 2013).However, substrates with high nitrogen levels, such as slaughter-house residues, pig and poultry manure, often result in inhibitionof the AD process, due to release of ammonia during their degrada-tion, which affects the stability and performance of the AD process

in full-scale reactors (Moestedt et al., 2016). Ammonium ion (NH4+)

and free ammonia (NH3) are the two primary forms of inorganicammonia nitrogen in aqueous solution (Chen et al., 2008). Themechanisms of ammonia inhibition in AD process have been thor-oughly studied and NH3 has been identified as the most toxic formof ammonia. In short, the passive diffusion of hydrophobic ammo-nia molecules into the microbes’ cells may cause potassium defi-ciency, intracellular pH changes, increase maintenance energyrequirements and suppress specific enzyme reactions (Sprott andPatel, 1986). The NH3/NH4

+ equilibrium is affected by the tempera-ture, the pH and the total ammonia (NH4

+ + NH3) concentration inAD process (Rajagopal et al., 2013). In detail, as the pH and/or







FEMS Microbiology Ecology, 91, 2015, fiv130

doi: 10.1093/femsec/fiv130Advance Access Publication Date: 21 October 2015Research Article

RESEARCH ARTICLE

Ammonia effect on hydrogenotrophic methanogensand syntrophic acetate-oxidizing bacteriaHan Wang, Ioannis A. Fotidis and Irini Angelidaki∗

Department of Environmental Engineering, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark∗Corresponding author: Department of Environmental Engineering, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.Tel: (+45) 45251429; Fax: (+45) 45933850; E-mail: [email protected] sentence summary: SAOB were more sensitive to high ammonia compared to the hydrogenotrophic methanogens tested. Thus, hydrogenotrophicmethanogens could be equally, if not more, tolerant to high ammonia levels compared to SAOB.Editor: Alfons Stams

ABSTRACT

Ammonia-rich substrates can cause inhibition on anaerobic digestion process. Syntrophic acetate-oxidizing bacteria (SAOB)and hydrogenotrophic methanogens are important for the ammonia inhibitory mechanism on anaerobic digestion. Theroles and interactions of SAOB and hydrogenotrophic methanogens to ammonia inhibition effect are still unclear. The aimof the current study was to determine the ammonia toxicity levels of various pure strains of SAOB and hydrogenotrophicmethanogens. Moreover, ammonia toxicity on the syntrophic-cultivated strains of SAOB and hydrogenotrophicmethanogens was tested. Thus, four hydrogenotrophic methanogens (i.e. Methanoculleus bourgensis, Methanobacteriumcongolense, Methanoculleu thermophilus and Methanothermobacter thermautotrophicus), two SAOB (i.e. Tepidanaerobacteracetatoxydans and Thermacetogenium phaeum) and their syntrophic cultivation were assessed under 0.26, 3, 5 and 7 g NH4

+-NL−1. The results showed that some hydrogenotrophic methanogens were equally, or in some cases, more tolerant to highammonia levels compared to SAOB. Furthermore, a mesophilic hydrogenotrophic methanogen was more sensitive toammonia toxicity compared to thermophilic methanogens tested in the study, which is contradicting to the general beliefthat thermophilic methanogens are more vulnerable to high ammonia loads compared to mesophilic. This unexpectedfinding underlines the fact that the complete knowledge of ammonia inhibition effect on hydrogenotrophic methanogens isstill absent.

Keywords: ammonia inhibition; anaerobic digestion; biogas; SAOB; syntrophic growth

INTRODUCTION

Anaerobic digestion is a biological treatment for organic wastesby which pollution control and renewable energy can be ob-tained at the same time. Specifically, anaerobic digestion is amultistep process consisting of four steps: hydrolysis, acidoge-nesis, acetogenesis and methanogenesis, which are performedby different groups of microorganisms (Angelidaki et al. 2011).However, substrates that contain high total ammonia (NH4

+ +NH3) levels can inhibit the anaerobic digestion process and re-sult in suboptimal biogas production (Fotidis et al. 2014). The

unionized form of ammonia (free ammonia) is considered asthe main toxic compound causing ammonia inhibition. Specifi-cally, Sprott and Patel (1986) and Gallert, Bauer andWinter (1998)reported that passive diffusion of free ammonia into the mi-crobes cells is causing proton imbalance, potassium deficiency,increase maintenance energy requirements and suppress spe-cific enzyme reactions. Total ammonia concentration, temper-ature and pH affect free ammonia concentration in anaerobicdigestion process (Chen, Cheng and Creamer 2008). Specifically,the shift from NH4

+ to NH3 is enhanced alongside the increaseof pH and temperature and results in increased toxicity on the

Received: 8 July 2015; Accepted: 16 October 2015C© FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]

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